Effect of recombinant protein expression on baculovirus budded virus structure and infectivity

重组蛋白表达对杆状病毒芽胞病毒结构和感染性的影响

• 批准号: ST/Y000498/1
• 负责人: Linda King
• 金额: $ 1.19万
• 依托单位: Oxford Brookes University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=ST%2FY000498%2F1
• 关键词: Effect; recombinant; protein; expression; baculovirus

Baculovirus expression vectors (BEVS) have become one of the most versatile and successful systems for the production of recombinant proteins. Frequently, they succeed in producing difficult targets where other systems have failed. Increasingly, they are employed to produce recombinant protein subunit vaccines, e.g. influenza, Covid-19 and several for animal diseases. They are also very useful for producing large, complex transmembrane proteins for drug discovery.The natural replication cycle of baculoviruses in insect cells involves early, late and very late phases of gene expression. Notably, the late phase sees the production of infectious virus particles that bud from the plasma membrane and are essential for virus dissemination in cell culture. Recombinant proteins are largely produced in the very late phase, however, some protein synthesis initiates in the late phase and if the target is cytotoxic this can have an impact on budded virus (BV) yield. This may reduce the infectious titre of virus by up to 10-fold. While this is not usually a problem in small scale tests to monitor recombinant proteins, it can have serious consequences in subsequent scale up work where large cultures of insect cells are infected with BEVS using high particle:cell ratios. A further issue is the production of BV stocks with high titres that may not be stable upon storage. Rather than maintaining a high titre for months, this period reduces to a few weeks of storage in a cold room. The cause(s) of the virus low titre and instability are unknown, apart from the link with recombinant protein production. We hypothesise that the structural integrity of BV is affected by the recombinant protein. However, BV structural data is very scarce due to limited access to state-of-the-art facilities, leading to a lack of in-depth understanding of these expression vectors and impacting on further BEVS development. With world-leading structural-characterisation facilities at STFC RAL and ISIS we can examine and investigate the structural integrity of recombinant BV with compromised infectious titre or stability on storage. The successful completion of this structural study will allow us to formulate intelligent design strategies for recombinant protein production that mitigate their effect on BV structure and infectivity. Thiswill allow projects that currently fail at the feasibilioty stage to progress to scale up and protein production for many different academic and commercial uses.

杆状病毒表达载体（BEVS）已经成为用于生产重组蛋白的最通用和最成功的系统之一。通常，他们成功地在其他系统失败的地方产生困难的目标。它们越来越多地用于生产重组蛋白亚单位疫苗，例如流感、Covid-19和几种用于动物疾病的疫苗。杆状病毒在昆虫细胞中的自然复制周期包括基因表达的早期、晚期和非常晚期。值得注意的是，晚期看到从质膜出芽的感染性病毒颗粒的产生，并且对于细胞培养物中的病毒传播是必需的。重组蛋白主要在非常晚期产生，然而，一些蛋白质合成在晚期启动，并且如果靶标具有细胞毒性，则这可能对出芽病毒（BV）产量产生影响。这可能使病毒的感染滴度降低多达10倍。虽然这在监测重组蛋白的小规模测试中通常不是问题，但在随后的放大工作中可能会产生严重后果，其中使用高颗粒：细胞比用BEVS感染昆虫细胞的大型培养物。另一个问题是生产具有高滴度的BV原液，其在储存时可能不稳定。而不是保持高滴度数月，这一时期减少到几个星期的储存在冷藏室。除了与重组蛋白生产有关外，病毒低滴度和不稳定性的原因尚不清楚。我们假设BV的结构完整性受到重组蛋白的影响。然而，由于最先进的设施有限，BV结构数据非常稀缺，导致对这些表达载体缺乏深入了解，并影响了BEVS的进一步发展。利用STFC RAL和ISIS的世界领先的结构表征设施，我们可以检查和研究重组BV的结构完整性，以及受损的感染滴度或储存稳定性。这项结构研究的成功完成将使我们能够制定重组蛋白生产的智能设计策略，以减轻其对BV结构和感染性的影响。这将使目前在可行性阶段失败的项目能够扩大规模，并将蛋白质生产用于许多不同的学术和商业用途。